Throughout a lifetime, a small population of self-renewing stem cells continuously replenishes cells and tissues of the body. Stem cell sources such as bone marrow (BM), mobilized peripheral blood (MPB), fetal liver, placenta, embryonic stem cells, and umbilical cord blood (UCB) all contain a variety of stem and progenitor cells (SPC) capable of reconstituting various cell lineages. These SPC remain quiescent until specific biological signals induce differentiation and proliferation. However, the specific signals are rarely known.
Hematopoiesis is the process of blood cell development from stem cells residing in the adult bone marrow (Bonner, et al., “The Blood and the Lymphoid Organs,” in Pathology (Rubin et al., eds., 1999), pp. 1051-1061). Hematopoietic stem cells (HSCs) in an adult typically reside in the bone marrow and the connective tissue filling up the cavities inside the long bones (breast bone, skull, hips, pelvic, ribs, and spine) of the body (Dexter and Spooncer (1987) Annu. Rev. Cell. Biol. 3:423; Verfaillie et al. (1999) 4:321), but have also been found in umbilical cord blood (UCB), mobilized peripheral blood, and placenta, and they have been derived from embryonic stem cells. The connective tissue consists of a complex network of solid cords separated by sinusoids. The cords are composed of stromal and hematopoietic cells, knitted together by an extensive and fertile extracellular matrice (ECM) (Bonner, et al., “The Blood and the Lymphoid Organs,” in Pathology (Rubin et al., eds., 1999), pp. 1051-1061). Arteries and capillaries within the bone provide blood and nutrients to the cells of the bone marrow.
Hematopoietic stem cells can be divided into four categories based on their ability to self-renew and commitment to a certain cell lineage: (i) pluripotential stem cells with unlimited self-renewal capacity; (ii) multipotential progenitor cells, also with unlimited self-renewing capacity, but committed to either lymphoid or non-lymphoid cell lineages; (iii) unipotential progenitor cells with limited self-renewal capacity and commitment to discrete blood cell lineages; and (iv) differentiated precursor cells with no self-renewing ability (Bonner, et al., “The Blood and the Lymphoid Organs,” in Pathology (Rubin et al., eds., 1999), pp. 1051-1061; and Cabrita et al. (2003) Trends Biotechnol. 21:233.)
Hematopoietic stem cells are commonly classified by the presence or absence of cell surface antigens (surface markers). CD34, which is the most commonly accepted marker for hematopoietic stem cells, is believed to be expressed on HSCs as well as on the majority of committed progenitors. See, for example, Yu et al. (1996) J. Formos. Med. Assoc. 95:281. Selection of CD34+ cells from bone marrow, peripheral blood, or cord blood will thus lead to a heterogeneous cell mixture containing not only primitive HSCs but also more mature, lineage specific progenitors. Id. CD34 expression on human hematopoietic stem cells is reversible. See, for example, Nolta et al. (2002) Leukemia 16(3):352-61. Human CD34+CD38− cells after long-term engraftment (7-18 months) in bnx mice lost expression of CD34 as they became deeply quiescent. Id. A CD45+CD34−Lineage− cell population recovered from the bone marrow of mice after long-term engraftment of CD34+CD38− cells was able to create colony-forming units contained long-term culture initiating cells (LTC-IC) and gave rise to multilineage engraftment with up-regulation of CD34 expression in secondary, immune-deficient mice. Id. CD34+CD38− cells are believed to be one of the most primitive populations of stem/progenitor cells that have been identified to date. See, for example, Nolta et al. (2002) Leukemia 16(3):352-61. Lack of, or very low level, expression of HLA-DR (human leukocyte antigen-locus DR) has been associated with extensive in vitro expansion capacity and in vivo stem cell function. See, for example, Yu et al. (1996) J. Formos. Med. Assoc. 95:281. An increase in HLA-DR expression on adult bone marrow or peripheral blood derived cells was found to correlate with lineage commitment. Id. In contrast, fetal as well as cord blood HSCs were reported to express HLA-DR. Id. Thy-1 expressed at low levels characterizes a subset of CD34+ cells with stem cell function. Id. CD133, also sometimes called AC133, is another cell surface marker commonly associated with hematopoietic stem cells.
Some researchers have attempted to expand stem cells ex vivo using defined cytokine cocktails with and without animal-derived serum and with and without cell feeder layers. See, for example, Dexter and Spooncer (1987) Annu. Rev. Cell. Biol. 3:423; Verfaillie et al. (1999) 4:321; Donovan and Gearhart (2001) Nature 414:92; Dexter et al. (1984) Blood Cells 10:315. It is widely accepted that using stroma-free, cytokine-supplemented cultures results in a large mature cell expansion. In fact, in most cases it is observed that the number of long-term culture initiating cells (LTC-IC) steadily decline indicating that expanded stem cells lose their developmental potential in culture, and that this potential eventually drops below the input level. See, for example, Yu et al. (1996) J. Formos. Med. Assoc. 95:281. Systems requiring animal-derived serum or exogenous feeder cells increase the risk of adverse immunogenicity reactions and the occurrence of infection in the recipient. Thus, there is a need in the art for chemically well-defined, such as cytokine-supplemented, cultures that sustain HSC growth over time, such that the amount of cells does not drop below the input level.
If stem cells capable of reconstituting particular lineages can be in vitro expanded and the developmental potential maintained, then therapies can be improved by decreasing the amount of donor material needed for therapy. Expanded SPC have the potential for use in stem cell transplantation in cancer therapy. In addition, the potential use of SPCs in other cellular therapies has been demonstrated, such as diabetes, heart disease, liver regeneration, and neurodegenerative diseases.
Another potential area of application for expanded SPCs is tissue engineering. A common problem encountered in tissue engineering applications, where culture-expanded cells can be used to create custom grafts of any size or shape to reconstitute, repair, and replace damaged or diseased tissues, is providing sufficient nutrients to cells residing inside solid engineered tissues exceeding a certain size. Certain SPCs, such as endothelial progenitor cells, have recently been shown to initiate neovascularization.
Neovascularization (formation of new blood vessels) can result from one of two distinct processes, vasculogenesis or angiogenesis. Vasculogenesis involves differentiation of endothelial progenitor cells (EPCs) into endothelial cells during embryogenesis. Angiogenesis entails the sprouting of capillaries from existing blood vessels. Commonly used strategies to induce neovascularization are focused on using controlled release of growth factors or DNA to promote angiogenesis, and only a few studies have attempted to utilize endothelial progenitor cells in combination with growth factor delivery. Importantly, stem cell culture systems would obviate the practical limitations associated with fresh donor material because stem cells can be frozen and then expanded prior to use. Thus, methods for the expansion of SPC are needed.
Megakaryocytes reside in the bone marrow and are the precursors to platelets. As compared to bone marrow (BM) and mobilized peripheral blood stem cell (mPBSC) transplants, engraftment to platelets is significantly delayed in cord blood stem cell transplants, i.e., 9 days for mPBSC as compared to anywhere from 56 to over 200 days. During this time, a patient is susceptible to bleeding and receives platelet transfusions to mitigate this problem. There is thus a need to shorten the time to platelet engraftment for patients receiving cord blood stem cell transplants to reduce the risk of bleeding, the need for platelet transfusions over an extended period of time, and ultimately to reduce the time of the hospital stay following the transplant.